Throughout this application, various references are referred to within parentheses. Disclosures of these publications in their entireties are hereby incorporated by reference into this application to more fully describe the state of the art to which this invention pertains. Full bibliographic citation for these references may be found at the end of this application, preceding the claims.
Terminal differentiation in human melanoma cells correlates with temporal changes in the expression of specific target genes. To define those genes that may be critical for this process a subtraction hybridization approach was used. cDNA libraries were constructed from actively proliferating HO-1 human melanoma cells (driver cDNA library) and cultures treated for various time periods with the combination of recombinant human fibroblast interferon (IFN-xcex2) and mezerein (MEZ) (temporally spaced tester cDNA library) that induces terminal differentiation (Jiang and Fisher, 1993). From these two cDNA libraries, an HO-1 IFN-xcex2+MEZ temporally spaced subtracted (TSS) cDNA library was constructed. Random screening of this TSS cDNA library identifies cDNAs that display differential expression as a function of induction of growth arrest and terminal differentiation, called melanoma differentiation associated (mda) genes. In the present study the properties of the novel mda-9 gene were analyzed. This cDNA encodes a unique protein of 298 amino acids with a predicted size of 32 to xcx9c34 kDa. Southern blotting analysis indicates that mda-9 is an evolutionary conserved gene. Tissue distribution analysis documents comparable expression in fifty human tissues, with slightly elevated expression in brain (putamen) and spleen (adult and fetal). Treatment of HO-1 human melanoma cells with IFN-xcex2+MEZ results in a biphasic induction of mda-9 with maximum expression 8 and 12 h post-treatment and reduced expression at 24 h. In terminally differentiated and irreversibly growth arrested human melanoma cells, the level of mda-9 mRNA is reduced. The suppression in mda-9 expression is not simply a function of growth inhibition, since treatment of HO-1 cells with interferons, including IFN-xcex2, leukocyte interferon (IFN-xcex1) or immune interferon (IFN-xcex3), elevates mda-9 expression even though they suppress growth. These studies demonstrate that subtraction hybridization using temporally spaced RNA samples, resulting in a TSS cDNA library, can identify genes, such as mda-9, that are down-regulated during terminal cell differentiation in human melanoma cells. Further studies are necessary to define the precise role of mda-9 in the process of terminal differentiation.
Cancer is a progressive disease characterized by both qualitative and quantitative changes in the phenotypes of evolving tumor cells (1-5). Although cancer can develop as a consequence of single or multiple genetic alterations, a common theme in carcinogenesis involves abnormal programs of differentiation (6-10). Attempts to exploit this defective differentiation process in cancer cells has led to the development of a therapeutic approach called xe2x80x98differentiation therapyxe2x80x99 (6-11). This strategy is based on the use of single or multiple agents that induce cancer cells to become more differentiated with a concomitant reduction or loss of growth potential (6-12). In order to utilize differentiation therapy as an effective clinical tool, further research is necessary to identify agents capable of efficiently inducing terminal differentiation in cancer cells without inducing nonspecific toxicity in normal cells. Additionally, the identification of genes that correlate with and may mediate terminal cell differentiation would represent valuable reagents for defining the molecular basis of terminal cell differentiation, for direct cancer therapeutic applications and for screening compounds for potential use in differentiation therapy (6-12).
In cultured human melanoma cells, the combination of IFN-xcex2+MEZ results in terminal cell differentiation and an irreversible loss of proliferative potential (11, 13, 14). In this model system, a single treatment for 24 h is sufficient to induce  greater than 95% terminal differentiation in cells subsequently grown for 72 h in the absence of inducers (14,15). The rapid induction of terminal differentiation in the vast majority of treated cancer cells makes this system amenable for defining those gene expression changes that occur during and that may mediate this process (11,12,16-19). To begin to address on a molecular level the question of growth control and terminal differentiation in human melanoma cells and to directly clone genes involved in these processes we developed and used an efficient subtraction hybridization protocol (16). This approach has resulted in the cloning of both known and novel cDNAs that are differentially regulated during growth suppression, reversible differentiation and terminal differentiation in human melanoma and other cancer cell types (16-20). The contents of U.S. Pat. No. 5,643,761, issued Jul. 1, 1997 to Fisher et al. entitled xe2x80x9cMethod for Generating a Subtracted cDNA Library and Uses of the Generated Libraryxe2x80x9d and of International Application PCT/US94/12160 filed Oct. 24, 1994, entitled xe2x80x9cMethod for Generating a Subtracted cDNA Library and Uses of the Generated Libraryxe2x80x9d which published May 4, 1995 as WO 95/11986 are hereby incorporated by reference.
In the present study, the properties of a novel mda-9 gene identified by subtraction hybridization were described. mda-9 is an evolutionary conserved gene that encodes a protein of xcx9c32 to xcx9c34 kDa without sequence homology to previously identified proteins. Expression of mda-9 is seen in fifty human tissues, with slightly elevated expression in brain (putamen) and spleen (adult and fetal). Induction of growth suppression and differentiation in human melanoma cells following exposure to IFN-xcex2+MEZ results in a decrease in mda-9 expression. These studies provide additional support for the hypothesis that induction of terminal differentiation and irreversible growth arrest in human melanoma cells involves multiple gene expression changes, including increases as well as decreases in the expression of specific target genes.
This invention provides a method for producing a temporally spaced subtracted cDNA library comprising: a) isolating temporally spaced RNAs from cells; b) generating cDNA inserts from the RNAs isolated from step (a); c) producing a temporally spaced cDNA library having clones containing the cDNA inserts generated from step (b); d) producing double stranded cDNA inserts from the temporally spaced cDNA library; e) denaturing the double stranded cDNA inserts; f) contacting the denatured double stranded cDNA inserts produced in step (e) with single-stranded DNAs from another cDNA library under conditions permitting hybridization of the single-stranded DNAs and the double-stranded cDNA inserts; g) separating the hybridized cDNA inserts from the unhybridized inserts; h) generating a cDNA library of the unhybridized inserts, thereby generating a temporally spaced subtracted cDNA library.
This invention further provides a temporally spaced subtracted library generated by the method for producing a temporally spaced subtracted cDNA library comprising: a) isolating temporally spaced RNAs from cells; b) generating cDNA inserts from the RNAs isolated from step (a); c) producing a temporally spaced cDNA library having clones containing the cDNA inserts generated from step (b); d) producing double stranded cDNA inserts from the temporally spaced cDNA library; e) denaturing the double stranded cDNA inserts; f) contacting the denatured double stranded cDNA inserts produced in step (e) with single-stranded DNAs from another cDNA library under conditions permitting hybridization of the single-stranded DNAs and the double-stranded cDNA inserts; g) separating the hybridized cDNA inserts from the unhybridized inserts; h) generating a cDNA library of the unhybridized inserts, thereby generating a temporally spaced subtracted cDNA library.
This invention provides a temporally spaced subtracted library generated by using HO-1 melanoma cells treated with IFN-xcex2 and MEZ in a temporally spaced manner at 2, 4, 8, 12, 24, and 48 hours and, wherein the single-stranded nucleic acid molecules are from another cDNA library of HO-1 melanoma cells.
This invention provides a method of identifying a melanoma differentiation associated gene comprising: a) generating probes from clones of the temporally spaced subtracted library generated by using HO-1 melanoma cells treated with IFN-xcex2 and MEZ in a temporally spaced manner at 2, 4, 8, 12, 24, and 48 hours and cells, wherein the single-stranded nucleic acid molecules are from another cDNA library of HO-1 melanoma cells; and b) hybridizing the probe with the total RNAs or mRNAs from HO-1 cells treated with IFN-xcex2 and MEZ and the total RNAs or mRNAs from untreated HO-1 cells, hybridization of the probe with the total RNAs or mRNAs from the treated HO-1 cell but altered [no, reduced, or enhanced] hybridization with the total RNAs or mRNA from untreated cells indicating that the clone from which the probe is generated carries a melanoma differentiation associated gene.
This invention provides a melanoma differentiation associated gene identified by the above described method of identifying a melanoma differentiation associated gene.
This invention provides a method of identifying temporally expressed genes from a single subtracted cDNA library, comprising: a) cloning the cDNAs from the temporally spaced subtracted cDNA library produced by the above described method for producing a temporally spaced subtracted cDNA library; b) hybridizing the clones obtained in step (a) with total RNAs isolated from control and with RNAs from differentiation-inducer treated cells, hybridization of the probe RNAs from differentiation-inducer treated cells, either enhanced or no or reduced hybridization with total RNA isolated from control cells indicating that the gene from which the probe was isolated is temporally expressed, thereby identifying temporally expressed genes from a single subtracted cDNA library.
This invention provides a temporally expressed gene identified by the above described method.
This invention provides an isolated mda-9 gene. This invention also provides an isolated nucleic acid having the nucleic acid sequence set forth in FIG. 7. This invention provides an isolated nucleic acid having the nucleic acid sequence set forth in FIG. 7, said nucleic acid encoding a human protein, wherein the encoded human protein is human mda-9. This invention also provides a human mda-9 protein having the amino acid sequence set forth in FIG. 7.
This invention provides a method for identifying a compound capable of inducing terminal differentiation in cancer cells comprising: a) incubating an appropriate concentration of the cancer cells with an appropriate concentration of the compound; b) measuring the expression of mda-9, the reduced expression of mda-9 gene indicating that the compound is capable of inducing terminal differentiation in cancer cells.
This invention provides a method for identifying a compound capable of inducing specific patterns of DNA damage caused by UV irradiation and gamma irradiation in human melanoma cells comprising: a) incubating an appropriate concentration of the human melanoma cells with an appropriate concentration of the compound; and b) measuring the expression of mda-9, the altered expression of mda-9 gene indicating that the compound is capable of inducing specific patterns of DNA damage caused by UV irradiation and gamma irradiation in human melanoma cells.
This invention provides a method for identifying a temporally expressed gene from cancer cells induced to undergo apoptosis by a chemotherapeutic agent, comprising: a) incubating an appropriate concentration of the cancer cells with an appropriate concentration of the chemotherapeutic agent; and b) measuring the expression of mda-9, the modified expression of mda-9 gene indicating that the compound is capable of inducing the cancer cells to undergo apoptosis.
This invention provides a method for identifying a compound capable of elevating mda-9 expression in cancer cells comprising: a) incubating an appropriate concentration of the cancer cells with an appropriate concentration of the compound; b) measuring the expression of mda-9 to determine whether the expression of the mda-9 gene is elevated.
This invention provides a method for detecting the presence of cytokines in a sample comprising a) contacting the sample with cancerous cells; b) measuring the expression of the mda-9 gene; c) determining whether the expression of the mda-9 gene is altered, the altered expression of the mda-9 gene in the cancerous cells indicating the presence of cytokines.
This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human mda-9 protein so as to prevent expression of the mRNA molecule. This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human mda-9 protein so as to prevent translation of the mRNA molecule.
This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the promoter of the isolated nucleic acid molecule of an mda-9 gene, wherein the encoded mda-9 protein is a human protein, thereby preventing mRNA transcription.
This invention provides an antisense oligonucleotide having a sequence capable of specifically hybridizing to the mRNA of the isolated nucleic acid molecule of an mda-9 gene, wherein the encoded mda-9 protein is a human protein, and capable of degrading the hybridized mRNA.
This invention provides a purified mda-9 protein. This invention provides a purified human mda-9 protein. This invention further provides a purified human mda-9 protein having an amino acid sequence as set forth in FIG. 7.
This invention provides an antibody directed to a purified mda-9 protein. This invention provides an antibody directed to a purified human mda-9 protein. This invention further provides an antibody directed to a purified human mda-9 protein having an amino acid sequence as set forth in FIG. 7. This invention further provides an antibody capable of specifically recognizing an mda-9 protein. In an embodiment of the invention, the antibody is capable of specifically recognizing a human mda-9 protein.
This invention provides a pharmaceutical composition comprising an amount of the antisense oligonucleotide having a sequence capable of specifically hybridizing to an mRNA molecule encoding a human mda-9 protein so as to prevent expression of the mRNA molecule, to prevent translation of the mRNA molecule, effective to prevent expression of a human mda-9 protein and a pharmaceutically acceptable carrier. This invention also provides a pharmaceutical composition comprising an amount of the antisense oligonucleotide having a sequence capable of specifically hybridizing to the promoter of the isolated nucleic acid molecule of an mda-9 gene, wherein the encoded mda-9 protein is a human protein, thereby preventing mRNA transcription, effective to prevent expression of a human mda-9 protein and a pharmaceutically acceptable carrier. This invention further provides a pharmaceutical composition comprising an amount of the antisense oligonucleotide having a sequence capable of specifically hybridizing to the mRNA of the isolated nucleic acid molecule of an mda-9 gene, wherein the encoded mda-9 protein is a human protein and capable of degrading the hybridized mRNA, effective to prevent expression of a human mda-9 protein and a pharmaceutically acceptable carrier.
This invention provides a method of treating melanoma in a subject by administering a pharmaceutical composition comprising an amount of any one of the above described antisense oligonucleotides effective to prevent expression of a human mda-9 protein and a pharmaceutically acceptable carrier, thereby treating melanoma in a subject.
This invention provides a method of administering a pharmaceutical composition comprising an amount of any one of the above described antisense oligonucleotides effective to prevent expression of a human mda-9 protein and a pharmaceutically acceptable carrier.
This invention provides a method of inhibiting expression of a mda-9 gene in a subject comprising introducing a vector containing a nucleic acid molecule which renders the mda-9 gene functionless into the subject under conditions permitting the inhibition of the expression of the mda-9 gene.
This invention provides a method of treating a cancer in a subject by administering a pharmaceutical composition comprising an effective amount of the antibody capable of specifically recognizing an mda-9 protein, thereby treating the cancer in a subject.
This invention provides a method of increasing the expression of mda-9 to inhibit cell growth comprising transfecting cells with an expression vector comprising an mda-9 gene insert to induce expression of mda-9 in cells thereby inhibiting growth of the cells.
This invention also provides a method of treating a cancer in a subject by increasing mda-9 expression in cancer cells of the subject to induce partial differentiation in the cancer cells by administering a pharmaceutical composition comprising a targeting vector and an agent which partially induces differentiation.
This invention further provides a method of treating a cancer in a subject by increasing mda-9 expression in cancer cells of the subject to suppress growth of the cancer cells by administering a pharmaceutical composition comprising a targeting vector and an agent which partially induces differentiation.
This invention provides a cell having an exogenous indicator gene under the control of the regulatory element of a mda-9 gene.
This invention provides a nucleic acid molecule comprising a sequence of the promoter of an mda-9 gene protein.